Bacterial vaginosis (BV) is the world's most prevalent vaginal infection, affecting hundreds of millions of reproductive-aged women every year. It is a polymicrobial condition that frequently recurs following successful treatment and is associated with HIV-1 infection, other sexually transmitted infections (STIs), and adverse reproductive health outcomes. A number of bacterial species are associated with BV. However, the exact etiology remains unknown. Innovative treatment and prevention approaches are needed to reduce the burden of disease caused by BV and its associated complications. Periodic presumptive treatment (PTT) is currently being evaluated as a novel strategy to reduce the incidence of vaginal infections, including BV. In May 2011, we began enrolling participants into a multi-site, randomized controlled trial to evaluate the effectiveness of PPT with intravaginal metronidazole 750mg plus miconazole 200mg vaginal suppositories versus matching placebo in reducing vaginal infections (Preventing Vaginal Infections [PVI] trial; DMID protocol #09-0070). Throughout the trial, we will archive vaginal fluid specimens for future testing of vaginal microbiota by molecular methods. The current proposal will utilize data and specimens collected as part of the PVI trial to assess the effects of PPT versus placebo on both cultivable and cultivation-resistant vaginal microbiota using cutting-edge molecular technologies. In Aim 1, we will use highly sensitive species-specific quantitative PCR assays to measure the effect of PPT on the detection and concentrations of bacterial species associated with vaginal health (L. crispatus, L. jensenii, and L. iners). Aim 2 will use similar laboratory methods to assess the effect of PPT on the detection and concentration of BV-associated bacterial species (BVAB1, BVAB2, BVAB3, Gardnerella vaginalis, Atopobium vaginae, Leptotrichia amnionii, and Megasphaera species). Aim 3 will utilize a complementary approach, employing broad range PCR with high throughput pyrosequencing to assess the impact of PPT on overall bacterial community composition and diversity in the vagina. Interventions that promote vaginal health by increasing the prevalence and quantity of Lactobacillus species and reducing the prevalence and quantity of species associated with BV could have a substantial impact on women's reproductive health. This proposal will generate unique and valuable information that will contribute to our overall understanding of the vaginal environment, and will inform future use of this PPT regimen as an intervention to reduce the risk of HIV-1, other STIs, and adverse reproductive health outcomes.